Na+/H+ exchangers (NHEs) mediate electroneutral, amiloride-sensitive exchange of Na+ and H+ across plasma membranes in most eukaryotic cells. In the mammalian kidney, this activity contributes to the maintenance of acid/base balance and NaCl homeostasis and has been described on both apical and/or basolateral surfaces of most segments of the nephron. Although the Na+/H+ exchanger located on the apical membrane (brush border) of the proximal tubule is sensitive to many factors including metabolic pH and hormones such as angiotensin II and parathyroid hormone the molecular mechanisms involved in regulation are not understood. This project will test the hypothesis that subcellular compartmentalization and molecular assembly represent critical aspects of the regulation of the renal brush border Na+/H+ exchange activity. Membrane fractionation studies in our laboratory have shown that the brush border Na+/H+ exchanger, NHE3, exists in both microvillar and non-microvillar membranes and that the relative level of the expression of NHE3 in these compartments shifts in relation to the acid/base status of the animal. Moreover, recent immunochemical studies have also shown that NHE3 exists in two distinct oligomeric states. One is a large (21S) complex with the brush border protein megalin and which is concentrated in dense, non-microvillar membranes, The other is a smaller (9.6S) form, not associated with megalin, and which is concentrated in the microvilli of the brush border. These data allow us to propose a model in which the Na+/H+ exchanger in regulated by mechanisms that include both the interaction with megalin and trafficking between distinct membrane compartments. Based upon these preliminary data we will describe at the molecular level the regulation of this important renal transport activity.